APPLICATION OF PROTON NMR-SPECTROSCOPY TO MEASUREMENT OF WHOLE-BODY RNA DEGRADATION RATES - EFFECTS OF SURGICAL STRESS IN HUMAN PATIENTS

The urinary catabolites, N-2,N-2-dimethylguanosine (DMG), pseudouridin e (PSU) and 7-methylguanine (m(7)-Gua) are formed from post-transcript ional methylation of RNA bases and are not reincorporated into RNA upo n its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species s ince DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and m (7)-Gua occurs in all RNA species. Conventional HPLC analysis has seve ral drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-fiel d H-1-nuclear magnetic resonance (H-1-NMR) spectroscopy. Urinary conce ntrations of these metabolites were found to be in agreement with prev iously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and rec onstitution) and was capable of the simultaneous determination of othe r relevant analytes such as creatinine. This technique was therefore a pplied to urine samples from patients who had undergone surgical stres s and insulin-like growth factor-I (IGF-I) therapy. Surgical stress in creased the excretion of DMG and m(7)-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compare d with controls but degradation rates of rRNA decreased by approx. 30% . However, injection of IGF-I (40 mu g/kg s.c.) had no significant eff ect on the excretion of these nucleosides. These data indicated that I GF-I therapy has no marked effects on RNA turnover following trauma. W e suggest that this technique can be applied to study of RNA metabolis m in any surgical or medical condition. Furthermore, since only 0.6 mi of urine is required, studies in neonates seem to be feasible.